Expressing humanized bacterial luciferase in stem cells: Moving beyond firefly luciferase to expand the informational capacity of animal models for regenerative medicine Project Summary This Small Business Technology Transfer (STTR) Phase II project proposes to develop complementary autonomously bioluminescent (autobioluminescent) in vitro stem cell lines and in vivo small animal model systems that enable the continuous, reagent-free, and real-time bioimaging of mesenchymal stem cell (MSC) localization, differentiation into adipocyte, chondrocyte, and osteocyte lineages, and persistence post- differentiation at the site of activation. These models will specifically address the National Institutes of Health's request for new techniques for non-invasive, long-term tracking of stem cell survivability, engraftment, and migration following in vivo implantation. By addressing this critical need for new methods capable of elucidating the mechanisms underlying how stem cells identify areas of dysfunction within the body, differentiate into the relevant tissues required to correct the malady, and persist in synergy with existing tissue to enable long term functionality, these tools will significantly improve the transition of regenerative medicine studies towards translational and clinical practice outcomes. The autobioluminescent MSCs developed by 490 BioTech under our Phase I effort demonstrated the ability to track MSC localization in vitro and in vivo similarly to existing optical imaging approaches, but with significantly reduced cost and personnel effort. Furthermore, these models also negated the need for sample destruction or the stressful and potentially influential injection of an activating chemical concurrent with imaging while simultaneously providing an uninterrupted stream of visual data over the lifetime of the reporter cell as it interacts with its environment and undergoes differentiation. In partnership with the University of Tennessee Medical Center, this proposal will expand upon these accomplishments to develop fully self-contained autobioluminescent MSC-based cellular models capable of specifically reporting on their differentiation into adipocyte, chondrocyte, and osteocyte lineages, and complementary small animal models harboring native MSCs genetically programmed to autonomously enact their reporter functionality only following differentiation into adipocyte, chondrocyte, or osteocyte lineages in response to wounding or exogenous stimulation. These models will overcome the primary technical hurdles encountered with all existing bioluminescent and fluorescent stem cells currently on the market from companies such as PerkinElmer, ThermoFisher/Life Technologies, Promega, and ~30 smaller specialized business entities in the U.S. alone, which comprise an estimated market value of at least $2B, with a predicated annual growth rate of 16-40%. We believe that the products developed in this effort will be capable of significantly improving the throughput and effectiveness of regenerative medicine studies and advancing our understanding of stem cell-based treatment efficiency and efficacy to improve both public health and consumer safety. The functional demonstrations and data gathered in this effort will position these models to thrive within this market and produce an immediate and significant impact on the field of regenerative medicine that will benefit the population at large.